Determinants of the quality of a humoral immune response, including the magnitude and isotype of antibody produced, and the extent of affinity maturation, remain poorly defined, limiting efforts of rational vaccine design. Whereas earlier studies had implicated innate immune recognition by Toll-like receptors (TLRs) of B cells in rapid lower affinity antibody responses, our recent studies have shown that innate recognition by TLR9 or TLR7 of B cells strongly enhances the magnitude and the efficacy of the germinal center (GC) response, which generates high quality antibodies. Moreover, this novel function of TLRs has been shown to be required for immune defense against several virus infections in mouse models. In addition, accumulating evidence has indicated that TLR7 and TLR9 are critical components required for the production of anti-nuclear antibodies in mouse models of systemic lupus erythematosus (SLE). Thus, a new pathway whereby TLRs promote high affinity GC antibody responses is likely to be important for immune defense against many viruses and for development of SLE. Recent studies have begun to define the cellular basis by which TLR recognition enhances the magnitude and quality of the IgG response, but the molecular mechanisms underlying this regulation are unknown, which is the topic of this application. Specific Aim 1 is designed to define the changes in mRNA and microRNA expression in GC B cells experiencing stimulation via their TLR9 compared to GC B cells responding to the same immunization but lacking a functional version of the TLR signaling component MyD88. Specific Aim 2 will address the hypothesis that key changes in gene expression are concentrated in subsets of GC B cells, namely GC B cells that are receiving selection signals from follicular helper T cells (TFH) and/or have a higher affinity for antigen. This issue will be assessed by microfluidic single cell analysis of gene expression profiles from GC B cells expressing a Myc-GFP reporter (resulting from productive interaction with TFH cells) or from GC B cells of defined affinity for antigen. Understanding the molecular mechanisms of this process will be useful for rational vaccine design and also many provide new targets for development of therapies to treat SLE.